Project A. Determine genetic susceptibility and immunopathological mechanisms contributing to idiopathic tooth root resorption, and delineate the role of IRF8 mutations in increased susceptibility to multiple idiopathic root resorption. Background: We noted that BSP KO mice exhibit a tooth root resorption phenotype. Based on this finding, we sought to identify human subjects exhibiting multiple idiopathic cervical root resorption, a familial pattern of periodontal disease with suggested genetic susceptibility. Following IRB approval from the University of Detroit Mercy School of Dentistry and NIH, dental/medical histories, x-rays, saliva samples, and extracted teeth were collected from a kindred (4 affected and 4 unaffected members) exhibiting multiple idiopathic cervical root resorption. On examination, the proband and the affected son and daughter exhibited severe root resorption of multiple teeth, with no other significant medical history. Micro-CT of exfoliated teeth revealed severe cervical root resorption distinct from tooth decay. Whole exome sequencing using saliva from affected and unaffected family members identified SNPs in ten candidate genes that co-segregated with the resorption phenotype, including a novel autosomal dominant missense mutation in the Interferon Regulatory Factor 8 (IRF8) gene. Primarily expressed in immune cells, IRF8 is a key regulator of inflammation and bone metabolism, and its repression mediates osteoclastogenesis by enhancing nuclear factor of activated T cells c1 (NFATc1) activity. The identified amino acid change (G388S) in IRF8 is localized to a highly conserved C-terminal motif, leading to altered serine phosphorylation motifs and phosphoserine binding domains, and is predicted to cause a large shift in 3D protein folding. These data suggest that the G388S mutation would impair IRF8 heterodimerization with other transcription factors including NFATc1, thereby producing overactive osteoclasts that target the periodontia. To study the effects of G388S mutation, human IRF8WT and mutant constructs were overexpressed in Irf8 KO macrophages and stimulated with M-CSF and RANKL. hIRF8G388S isoform promoted increased osteoclast differentiation, resorption pits, and osteoclast-gene expression. Furthermore, compared to hIRF8WT, the mutant hIRF8G388S isoform failed to inhibit NFATc1-dependent transcriptional activation of cathepsin-K reporter. RNA-seq analyses revealed that hIRF8G388S mutation enhanced global expression of osteoclast-specific genes, consistent with the transcripts noted from Irf8/ cells, suggesting that the increased osteoclastogenesis noted in both Irf8/ and hIRF8G388S cultures is a consequence of impaired IRF8 function. These results are planned to be submitted for peer review in Sept. 2018. Ongoing: In collaboration with Drs. Ozato and Holland, we are characterizing the periodontal phenotype in Irf8 KO mice and in patients with IRF8 mutations. We are generating IRF8 knock-in mice containing G388S mutation and an Irf8 conditional knock-out using macrophage-specific Cre. We plan to perform skeletal phenotype characterization and osteoclast experiments using these mice/cell cultures. Furthermore, we plan to perform RNA-seq and ChIP-seq on cell cultures from these animals to profile differentially regulated genes and IRF8 mutant binding sites. Project B. Disorders of mineralization: In collaboration with NIDCR clinical researchers and other IC clinicians, we have been examining individuals with mineralized tissue metabolism disorders for alterations in tissues/cells of the DOC complex. 1. Mutations in key regulators of Pi/PPi. Mineralization of skeleton and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. We examined the effect of decreased PPi on development and maturation of teeth in human subjects (4) with generalized arterial calcification of infancy (GACI), who harbor loss-of-function mutations in the ENPP1 gene. Three of the four subjects reported a history of infraocclusion or over-retained primary teeth or poor orthodontic tooth movement, suggesting altered mineral metabolism as a contributing factor. All subjects presented radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. Micro-CT analyses of extracted primary teeth from two GACI subjects revealed marked increase cervical cementum thickness and density vs. age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 knock-out mouse molars revealed a marked increase in acellular cementum thickness and volume. Collectively, these findings report a novel dental phenotype in GACI and further support our hypothesis that Pi/PPi modulation is as a key mechanism for regulating cementogenesis across species. Thumbigere-Math V et al., JDR, 2018. Ongoing: We are examining patients with Pi/PPi disorders at NIH CRC and are obtaining teeth when extractions are required. Project C. Chediak-Higashi Syndrome (CHS): Individuals with CHS, a rare autosomal recessive disease caused by mutations in the gene encoding lysosomal trafficking regulator, are immunodeficient, resulting in increased susceptibility to infections impacting several tissues, including oral tissues. In our ongoing collaboration with Dr. Inrone (NIH) and Drs. Nociti and Kantovitz from the State University of Campinas Brazil, seven CHS-diagnosed individuals were examined regarding their oral status in the NIH clinic, and gingival fibroblasts. Data from qPCR and multiplex protein analyses suggest that gingival fibroblasts from atypical patients exhibit an altered response to LPS suggestive of a more robust pro-inflammatory response to dental biofilm, and that higher levels of TLR-4 in atypical CHS GF play a key role in a hyperactive periodontal response to gram-negative bacteria. The Brazil group is now focusing on membranome analysis of gingival fibroblasts obtained from subjects with CHS. Thumbigere-Math et al., JDR CTR 2018.